Effects Of Growth Differentiation Factor 6 On Differentiation Of Stem Cells Derived From Rat Nucleus Pulposus Tissue

Background:Degenerative disc disease(DDD)is a prevalent disorder in modern society.Current therapies involve conservative symptomatic pain relief or end-stage surgical treatments.However,these therapies are relatively unsuccessful in preventing or reversing degeneration of intervertebral disk.Many reserchers have demonstrated the existence of nucleus pulposus(NP)-derived mesenchymal stem cells(MSCS).They are more adaptable to the harsh intervertebral disc(IVD)microenvironment than other types of stem cells are.Recently,some studies have reported that GDF6 improves differentiation of BM-MSCS to NP-like cells.However,few research reports about NP-derived MSCS differentiation into NP-like cells in rats.Thus,we harvested NPSCs from nucleus pulposus tissue of SD rats and stimulated with GDF6.the purpose of this work was to investigate the influence of GDF6 on differentiation of NP-derived MSCs into NP-like cells and to provide a new idea for patients who are suffering from DDD.Objective:To establish the model of rat marrow mesenchymal stem cells in vitro,and to observe the cytological characteristics and biological characteristics of rat nucleus pulposus mesenchymal stem cells.To study the effect of growth factor GDF6 on the differentiation of mesenchymal stem cells in rat nucleus pulposus.Methods:1.The nucleus pulposus tissue of the rat's tail was removed in sterile condition,and the culture of the nucleus pulposus of intervertebral disc was digested and separated by type II collagenase,subsequently cells were subcultured following by digestion with trypsin.2.The analysis of NP-derived MSCs surface-antigen CD105?CD90?CD34 and CD45 were done by flow cytometry.Additionally,the expression of classic and novel NP-derived MSCs marker genes SOX2 and Nanog were detected by real-time polymerase chain reaction(RT-PCR).3.It was divided two groups according to culture media consisting: group A: Standard cartilage induction medium(without TGF-beta 3)as control group;group B: Standard cartilage induction medium(without TGF-beta 3)+GDF6(100ng/ml)as experimental group.After 14 days of culture,the expression of KRT8,KRT18 and KRT19 genes were detected by fluorescence quantitative PCR.Results:Cells isolated from rat nucleus pulposus could form the sunflower-like colonies and exhibit clone-like growth.After 3 weeks the cell fusion can reach 90%.After subculture,the cell proliferation was accelerated,and the cells were spindle shaped.The markers of cell surface antigens were identified by flow cytometry,the results showed that it was positive for CD105 and CD90,negative for CD34 and CD45 in the cells of experimental group.Fluorescence quantitative PCR results demonstrated that the expression of KRT8?KRT18?KRT19 genes were significant higher in GDF6 induced group compared with control.(P<0.05)Conclusions:Firstly,we successfully isolated and cultured NP-derived MSCs from Nucleus pulposus tissue of SD rats.Secondly,our study indicated that GDF6 may be an optimal growth factor to drive NP-MSCs differentiation toward NP-like cells and provide theoretical basis for the treatment of intervertebral disc degeneration.